Although pathological fibrin deposition is a common feature of many life threatening vascular disorders, the underlying mechanism(s) remain obscure. It most certainly reflects an imbalance between the coagulation and fibrinolytic systems. The primary hypothesis of this proposal is that this imbalance results, in part, from local fibrinolytic shutdown due to the accumulation of type 1 plasminogen activator inhibitor (PAI-1) at sites of vascular injury since PAI-1 specifically binds to vitronectin (Vn) in the exposed extracellular matrix and to vimentin-type intermediate filaments in damaged cells. We have localized the high affinity PAI-1 binding site in Vn to the somatomedin B (SMB) domain. The general objectives of this proposal are to develop initial information about the structure, function and biosynthesis of SMB, Vn and other PAI-1 binding proteins (PAI-1 BPs) in vivo, and to establish their importance in human vascular disease. These goals will be pursued in 6 separate Specific Aims. In Specific Aim 1, we will define the minimal sequence in SMB required for PAI-1 binding using recombinant DNA technology to systematically develop deletion and substitution mutations through this region. The ability of these mutant proteins to bind and to stabilize PAI-1 and compete with native SMB and Vn for PAI-1 binding will be assessed. These results will be complemented by studies to identify the cross-linking site for PAI-1 in SMB and the epitopes recognized by inhibitory monoclonal antibodies (MABs), and by using synthetic peptides. Similar approaches will be employed to define the SMB-binding domain in PAI-1 (Aim 4). In Aim 2, we will crystallize SMB and determine its 3- dimensional structure by X-ray diffraction. Although free SMB has been detected in serum, its origin remains controversial. We will use available MABs to develop immunological assays that distinguish between free SMB and the SMB domain of Vn, and employ them to determine the concentration of free SMB in fractionated plasma, and in serum, tissues and cultured cells (Aim 3). If present, we will study the interaction between circulating SMB and PAI-1. Although Vn is produced primarily by the liver, Vn antigen has been detected in other tissues including the fibrous plaques of atherosclerotic arteries. However, the generality of this latter observation and the origin of Vn antigen (plasma vs local biosynthesis) remains to be determined. In Aim 5, we will examine normal mouse tissues and available normal and diseased human vascular tissue by immunological assays, competitive PCR, and in situ hybridization to systematically verify these observations to determine the relative levels of Vn mRNA present. Finally, in Aim 6 we will employ a variety of techniques to screen for and characterize new PAI-1 BPs in tissue since PAI-1 frequently accumulates in a pattern distinctly different from that of Vn. These studies will not only provide novel information about the structure and in vivo function of SMB and Vn, but will also begin to define the importance of tissue-specific PAI-1 BPs for the development of a local prothrombotic state.